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Rainer Cramer and Reiner Westermeier (eds.), Difference Gel Electrophoresis (DIGE): Methods and Protocols, Methods in Molecular Biology, vol. 854, DOI 10.1007/978-1-61779-573-2_1, © Springer Science+Business Media, LLC 2012

Chapter 1

DIGE: Past and Future

Jonathan S. Minden

Abstract

This chapter provides a brief historical perspective of the development of difference gel electrophoresis, from its inception to commercialization and beyond.

Key words: Difference gel electrophoresis , Historical perspective

As the inventor of difference gel electrophoresis (DIGE), I was asked to contribute a historical perspective of this method. This story is similar to other inventions where something was created to solve an unmet need. In the case of DIGE, I wanted to understand protein changes in different mutant backgrounds. The unusual element of this story is that many of the ancillary tools needed to make the invention work did not exist when the idea was fi rst hatched. It took more than a dozen years before these other technologies progressed far enough to make the entire process feasible. In this chapter, I will recount the history of the invention of DIGE—a humbling exercise that underscores my naiveté. As with any method, there are always ways to enhance and improve it. While the chemistry of DIGE is fairly well established and stable, the ancillary tools are continually being improved. However, there are persistent technological issues that will need to be resolved. In addition to the historical past of DIGE, I will outline some of the future challenges I see for DIGE.

1. Introduction

4 J.S. Minden

DIGE (which my lab pronounces as dee-gay, owing to the Turkish heritage of Mustafa Ünlü, the graduate student who synthesized the fi rst pair of DIGE dyes) was initially conceived when I was a fi rst year graduate student at Albert Einstein College of Medicine. I was tasked with analyzing a set of Dictyostelium discoideum temperature-sensitive, motility mutants. In 1981, the Dictyostelium genome was not known, and there were very few genetics tools for identifying the affected genes. Two-dimensional gel electrophoresis (2DE), which was only about 6 years old at the time, seemed like the best route for discovering the biochemical changes associated with these mutations. I wanted to compare these cells at the per-missive and restrictive temperatures. After a couple of abortive tries at running parallel 2DE gels, I realized that an internal control was needed. This inspiration probably came from one of the graduate classes I was taking at the time. The idea was to differentially label the two samples in separate reactions and then combine the two reactions so that they could be run on the same 2DE gel. Getting this idea to work presented several problems: what sort of tags should be used, how will the tags be attached to the proteins, will the tags effect how the proteins run on 2DE gels, how would the tags be detected…?

Proteins could be labeled metabolically in vivo or chemically in vitro. I wanted to make a versatile, user-friendly method that could be used on any source of protein. Since only a small number of model organisms are amenable to metabolic labeling schemes, chemical labeling after cell lysis seemed to be the most universal approach. Should the tag be radioactive, colored, or fl uorescent? Radioactivity was rejected because autoradiography often requires very long exposures and discrimination between radionuclides can be diffi cult. Colored tags would only work for very abundant proteins since color detection requires considerable amounts of absorbing mate-rial, particular in gels where the path length is 1–2 mm. Fluorescence seemed like the best choice since one can synthesize compounds with very different fl uorescent spectra for easy discrimination. Also, one can detect minute amounts of fl uorescent material, which I learned from the fl uorescence microscopy we were doing in the cell biology lab in which I was working.

I had no idea what fl uorescent molecules to use for DIGE. I poured through chemical catalogs and books on fl uorescent dyes, but none of the existing fl uorescent compounds really fi t the bill. A key point in selecting the right pair of dyes was that the dyes should have the same charge as each other and they should have similar masses. Another issue was that the dyes should be pH insensitive since they

2. The Past

2.1. The Conception of DIGE

2.2. What Sort of Tags Should Be Used?

2.3. The Selection of Fluorescent Labels

51 DIGE: Past and Future

will experience a broad pH range during isoelectric focusing. This latter consideration eliminated fl uorescein, the most popular fl uo-rescent dye of the day. The dyes should not change the charge of the protein to which they were attached, which raised the issue of how to attach the dyes to the proteins. The two most reactive amino acid residues are lysine and cysteine. Lysine’s primary amine provided an easy route to coupling the dye to the protein, but the coupling process eliminates lysine’s positive charge. This meant that the DIGE dyes had to have an amino group like lysine or an intrinsic positive charge. Given the p K a of the e -amino group of lysine and the 3–10 pH range of isoelectric focusing, a quaternary amine was a reasonable substitute for a primary amine. Alternatively, one could couple the DIGE dyes via cysteine residues. At that point in time, it was not known what fraction of proteins had at least one cysteine, and I was frankly unfamiliar with the coupling chemistry, so I put cysteine labeling on the backburner.

Since the literature search did not yield suitable DIGE dyes, I tried to design and synthesize my own dyes, but my chemistry knowl-edge was too limited. So I sent a letter to Kodak asking for help synthesizing the dyes. I got a nice letter back from their lawyers saying that they would help as long as I signed a form releasing my invention rights to Kodak. This was before the biotechnology industry took hold, and academic institutions rarely had technology transfer offi ces. I was in uncharted territory. This also coincided with my changing research advisors and a major shift in my research direction. Consequently, I left the idea behind for 10 years, during this period I completed my PhD in DNA replication and my post-doctoral training in cell and developmental biology. In retrospect, this hiatus was a good thing. In 1981, fl uorescence imaging sys-tems were too insensitive, and protein sequencing was only done by Edman sequencing—mass spectrometric protein identifi cation was only in its infancy.

In 1991, I accepted a faculty position at Carnegie Mellon University because of its reputation for interdisciplinarity and entrepreneurship. My lab primarily studies Drosophila embryo development, particularly how cells change shape during develop-ment. We were studying a specifi c cell shape change that had been extensively analyzed by genetic dissection. I was puzzled by the fact that none of the genetically identifi ed genes required for this cell shape altering process involved the cytoskeleton or its regula-tors. I reasoned that cytoskeleton was required for so many cellular functions that mutations in these genes would prevent the embryo from developing far enough. But I was certain that changes in the cytoskeleton and other proteins must occur during this process. This led me to dust off my old notebooks and reinvestigate DIGE. An equally important impetus for revisiting DIGE was that my lab was around the corner from Alan Waggoner’s, the father of cyanine

2.4. The Synthesis of the First DIGE Dyes

6 J.S. Minden

(Cy) dyes. Alan and I went to lunch one day and I recounted the four criteria for designing DIGE dyes. He said “oh, that’s easy” and immediately drew two structures on a napkin. True to CMU’s open-door, interdisciplinary policy, I recruited a fi rst year graduate student, Mustafa Ünlü, from the Chemistry Department. The fi rst pair of DIGE dyes, Cy3-NHS and Cy5-NHS, were synthesized in a few months. These are lysine-reactive dyes that have a net posi-tive charge and differ in molecular weight by 2 Da.

Since there were no fl uorescent-gel imagers that detected Cy3 and Cy5 at that time, we had to build our own imager. The very fi rst imager was constructed from a 12-bit, cooled CCD camera mounted on a darkroom enlarger stand. The light source was a 35-mm slide projector mounted on a repurposed rail from another darkroom enlarger stand, and the fl uorescent fi lters were mounted in a manual fi lter turret. This primitive imager was housed in a darkened room. The initial images were encouraging, but needless to say this was not a very light-tight arrangement. The next-generation imager was made more light-tight by building the imager around an IKEA cabinet with holes cut in the top and sides for the CCD camera and illuminator, respectively. This imager was suffi cient to begin optimizing the protein labeling reaction. Our initial plan was to saturation label all lysine residues of all proteins in a cellular extract. This gave very poor results since the proteins tended to precipitate. In retrospect, it was not a good idea to saturation label lysine as this addition would increase the protein mass by about 25%. Instead, substoichiometric labeling turned out to be the best way to maintain protein solubility and avoid size hetero-geneity due to multiple dyes molecules bound. In substoichiometric (or minimal) labeling, about 2–3% of all lysine residues are labeled. This translates to about 5% of all proteins having a single fl uorescent dye bound, while the rest are unlabeled. This set of innovations led to the fi rst published report on DIGE ( 1 ) .

A great deal of my lab’s efforts was assisted by Lans Taylor and Alan Waggoner’s Center for Light Microscopy and Biotechnology, an NSF Science Technology Center (STC). Lans and Alan had spun off several of their inventions into companies that were even-tually purchased by larger companies. Amersham, plc (now part of GE Healthcare) was interested in licensing the rights to the STC’s suite of cyanine dyes, including the DIGE dyes, which Lans and Alan negotiated masterfully. Working with Amersham to commercialize DIGE was an important learning experience. They were meticulous in establishing a robust, reliable protocol. Since the initial develop-ment of Cy3-NHS and Cy5-NHS, Amersham/GE has introduced Cy2-NHS for three-color minimal labeling and Cy3-mal and Cy5-mal for saturation labeling of cysteines. Amersham/GE also developed fl uorescent-gel imaging systems and image analysis

2.5. The First DIGE Experiments

2.6. Taking DIGE to Commercialization

71 DIGE: Past and Future

software dedicated to DIGE proteomics. These reagents and tools now make DIGE one of the most versatile and sensitive comparative proteomics methods currently available. This volume is a testament to the broad applicability and robustness of DIGE.

The goal of comparative proteomics is to discover protein changes between cells and tissues under a variety of conditions and circum-stances. Implicit in this goal is the desire to detect all protein species within the proteome. Given the chemical complexity of the proteome and its large number of different protein species, sepa-rating the proteome into discrete entities is a virtually impossible task, but we are obligated to do as best we can.

Historically, conventional 2DE has had diffi culty in resolving proteins that are very large (>250,000 Da) or very basic (>9.5 p I , many of which are ribosomal proteins). Mass spectrometry-centric methods also have diffi culties with proteins outside these ranges. Fortunately, these proteins represent a very minor component of the proteome. 2DE also has diffi culty resolving integral membrane proteins. This may be due to their hydrophobicity, which causes protein aggregation, and glycosylation, which causes heterogene-ity in mass and charge so that membrane proteins do not appear as discrete spots. Important strides in improving 2DE’s capacity to resolve membrane proteins have been made by methods such as introducing novel detergents, lipid removal, and deglycosylation ( 2– 4 ) . Finally, concern has been raised about protein overlap or comigration on 2DE gels. The development of narrow pH-range isoelectric focusing gels and very large-format 2DE gels has greatly improved the resolution of 2DE gels where over 10,000 protein species are now detectable ( 5, 6 ) . These improvements have sig-nifi cantly increased the resolving power of 2DE. One can expect even further advances since many laboratories continue to study new ways to increase the resolution of 2DE. These efforts have improved resolution relative to protein mass and p I , but compara-tively little has been done to improve resolution relative to protein abundance.

Proteins exist in cells over an approximately 10 5 -fold concen-tration range, while in serum the concentration range is on the order of tens of millions fold. Currently, DIGE imagers are capable of detecting proteins over a 20,000-fold concentration range. In contrast, conventional mass spectrometers have a dynamic range of about 1,000-fold. There is a clear need to improve the sensitivity and dynamic range of DIGE fl uorescent-gel imagers. I am heart-ened by the fact that technology for detecting single-molecule fl uorescence already exists. Hence, it should be possible to build

3. The Future

8 J.S. Minden

the next-generation fl uorescence imager that has the requisite dynamic range to detect proteins over a million-fold concentration range. Once this dynamic range goal is achieved, the next goal will be to isolate suffi cient quantities of protein from these very rare protein spots to be identifi ed by mass spectrometry. I am confi dent that these methods are within our reach. Thus, I feel that the future for DIGE is very bright (please forgive the pun).

Acknowledgments

The development of DIGE would not have been possible without the efforts and dedication of my students and staff (Mustafa Ünlü, Liz Morgan, Chris Lacenere, Surya Viswanathan, Lei Gong, Mamta Puri, Anupam Goyal, and Susan Down).

References

1. Ünlü, M., Morgan, M. E., and Minden, J. S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts, Electrophoresis 18 , 2071–2077.

2. Helling, S., Schmitt, E., Joppich, C., Schulenborg, T., Mullner, S., Felske-Muller, S., Wiebringhaus, T., Becker, G., Linsenmann, G., Sitek, B., Lutter, P., Meyer, H. E., and Marcus, K. (2006) 2-D differential membrane proteome analysis of scarce protein samples, Proteomics 6 , 4506–4513.

3. Comunale, M. A., Mattu, T. S., Lowman, M. A., Evans, A. A., London, W. T., Semmes, O. J., Ward, M., Drake, R., Romano, P. R., Steel, L. F., Block, T. M., and Mehta, A. (2004) Comparative proteomic analysis of de-N-glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status, Proteomics 4 , 826–838.

4. Ruan, Y., and Wan, M. (2007) An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE, Electrophoresis 28 , 3333–3340.

5. Han, M. J., Herlyn, M., Fisher, A. B., and Speicher, D. W. (2008) Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts, Electrophoresis 29 , 695–705.

6. Sitek, B., Sipos, B., Pfeiffer, K., Grzendowski, M., Poschmann, G., Hawranke, E., Koper, K., Kloppel, G., Meyer, H. E., and Stuhler, K. (2008) Establishment of “one-piece” large-gel 2-DE for high-resolution analysis of small amounts of sample using difference gel electro-phoresis saturation labelling, Anal Bioanal Chem 391 , 361–365.